The purpose of the proposed study is to determine the heat- and x-ray-induced changes that occur in nuclei and how such changes inhibit nuclear function. Following hyperthermia increases in nuclear protein content appear to correlate with cell killing, disruption of DNA synthesis and DNA repair. The results of these proposed studies will determine if heat-induced excess nuclear proteins disrupt these nuclear functions by alteration of enzymatic access to DNA or the DNA available for supercoiling changes. Additional experiments will correlate the presence and removal of excess nuclear proteins with the onset of heat-shock protein accumulation and the resumption of cell progression through the cell cycle. Proteins will be separated for nuclei from heated and control cells and analyzed by polyacrylamide gel electrophoresis. DNA supercoiling will be detected from the response of DNA in increasing concentrations of intercalating agents. Changes in the distribution of cells throughout the cell cycle will be measured using flow cytometry. These same techniques will be used to determine the role of nuclear protein changes during the X-ray-induced G2 block. The synthesis of nuclear proteins in G2 cells will be monitored by labeling with radioactive amino acids at various time intervals after irradiation. In addition changes in DNA supercoiling following x-irradiation will be monitored. These studies will delineate the mechanisms by which hyperthermia and x-irradiation disrupt cellular function. Since both of these agents are used in cancer therapy, the understanding of the above phenomena will contribute to the biological rationale for the clinical use of heat and radiation.